Influence  of  Certain  Amino  Acids 

upon  the  Enzymic  Hydrolysis 

of  Starch 


DISSERTATION 

Submitted  in  partial  fulfillment  of  the  requirements  for  the  degree 
of  Doctor  of  Philosophy  in  the  Faculty  of  Pure  Science,  Columbia 
University. 

By 
FLORENCE  WALKER,  A.B.,  A.M. 


NEW  YORK 
1922 


Influence  of  Certain  Amino  Acids 

upon  the  Enzymic  Hydrolysis 

of  Starch 


DISSERTATION 

Submitted  in  partial  fulfillment  of  the  requirements  for  the  degree 
of  Doctor  of  Philosophy  in  the  Faculty  of  Pure  Science,  Columbia 
University. 

By 

FLORENCE  WALKER,  A.B.,  A.M. 
N 


NEW  YORK 
1922 


ACKNOWLEDGMENT 

This  investigation  is  a  continuation  of  the  study 
of  amylases  begun  by  Professor  H.  C.  Sherman 
in  1907.  The  author  is  greatly  indebted  to  Pro- 
fessor Sherman  for  many  helpful  suggestions  re- 
ceived from  him  during  the  course  of  this  work. 


INFLUENCE  OF  CERTAIN  AMINO  ACIDS 

UPON  THE  ENZYMIC  HYDROLYSIS 

OF  STARCH 

In  1893  Effront1  stated  that  asparagine  accelerates  the  hydrolysis 
of  starch  by  malt  and  taka-diastase.  A  few  years  later2  he  obtained 
similar  activation  by  addition  of  certain  proteins  and  of  a  boiled 
cold  water  extract  of  barley.  In  1904  he  reported3  that  asparagine 
aspartic  acid,  hippuric  acid,  creatin,  creatinin  and  the  peptones  in- 
creased the  action  of  malt  extract,  while  succinamide,  the  amines 
and  their  salts,  and  acid  amides  generally  act  unfavorably.  This  he 
found  for  several  starches  of  different  origin.  He  also  found,  how- 
ever, that  the  more  favorable  the  conditions  for  the  production  of 
an  optimum  amount  of  sugar,  the  less  marked  is  the  effect  of  the 
amino  acids.  * 

Ford*,  working  with  malt,  found  asparagine  to  be  without  effect 
on  the  activity  of  the  enzyme.  The  apparent  activation  by  amino 
acids  and  acid  salts  obtained  by  other  investigators,  he  ascribes  to 
the  neutralization  by  these  compounds  of  alkaline  impurities  in  the 
starch. 

According  to  Terroine  and  Weill5  saccharification  by  pancreatic 
juice  is  greatly  accelerated  by  alanine,  glycine,  leucine,  valine,  histi- 
dine,  arginine,  tyrosine,  phenylalanine,  aspartic  acid  and  glutamic 
acid.  The  activating  power  which  they  found  the  digestion  products 
of  protein  to  possess  is,  they  think,  most  probably  due  to  the  amino 
acids  formed. 

More  recently  Rockwood6  has  investigated  the  effects  of  nitro- 
geneous  substances  on  the  hydrolysis  of  corn  starch  by  saliva  and 
concludes  that  glycine,  tyrosine,  anthranilic  acid  and  its  meta  and 
para  isomers,  aspartic  acid,  hippuric  acid,  proteins  (serum  albumin 
and  gelatin)  and  amines  of  the  methane  series  increase  saccharifica- 
tion, whereas  the  amides  (acetamide,  propionamide  and  urea),  sul- 
phanilic  acid,  asparagine,  succinamide  and  succinimide  show  no  such 

*Mon.   sci.,  41,  266    (1893). 
*Compt.   rendu.,  120,   1281    (1895). 

'Bull.  soc.  chim.,  [3]  31,  1230  (1904);  Mon.  sci.,  [4]  18,  561  (1904);  Compt.  rendu. 
soc.  biol.,  57,  234  (1904). 

4J.   Soc.   Chem.   Ind.,  23,   414   (1904). 

5  Compt.   rendu.   soc.   biol.,  72,   542    (1912). 

"Jour.  Amer.  Chem.  Soc.  39,  2745   (1917).  .    .. 


478719 


4  Influence   of   Certain   Amino   Acids 

effect.     He  also  found  that  glycine  and  aspartic  acid  activate  pan- 
creatic extract. 

Because  of  the  lack  of  agreement  among  previous  workers  as  to 
whether  amino  acids  do  or  do  not  activate  amylases,  and  in  view  of 
the  fact  that  past  investigations  have  seldom  covered  more  than  one 
enzyme,  a  systematic  study  of  the  influence  of  amino  acids  upon  the 
action  of  several  amylases,  in  purified  as  well  as  in  their  natural  or 
commercial  forms,  seemed  desirable.  An  attempt  has  been  made 
in  the  present  work  to  throw  some  light  on  the  manner  in  which  the 
amino  acids  act. 

EXPERIMENTAL 

Apparatus  and  Materials. — The  glassware  was  of  the  best  quality, 
carefully  selected  and  when  not  in  use  was  kept  filled  with  water. 
Just  before  using,  it  was  washed  with  soap,  rinsed  ten  times  with 
tap  water,  three  times  with  ordinary  and  once  with  triple  distilled 
water.  A  Freas  thermostat  bath  with  a  variation  of  +  0.005°  was 
used  for  all  digestions  at  40° C.  Digestions  at  other  temperatures 
were  carried  out  in  a  small  bath  kept  constant  within  +0.15°  by 
means  of  an  Ostwald  regulator. 

Merck's  "soluble"  starch  according  to  Lintner  purified  by  washing 
9  times  with  ordinary  distilled  and  6  times  with  triple  distilled  water 
has  in  all  cases  been  used  as  substrate.  The  salts  employed  as 
activators  were  C.  P.  recrystallyzed  several  times.  The  amylase 
preparations  and  other  enzyme  containing  materials  tested  were 
(1)  pancreatic  amylase  preparations  Nos.  58,  59,  60,  77B,  81B  and 
11B,  (2)  commercial  pancreatin  No.  8,  (3)  malt  amylase  preparation 
No.  155,  (4)  malt  extract,  (5)  aspergillus  amylase  preparations 
Nos.  22,  22b,  and  23,  (6)  commercial  takadiastase,  and  (7)  fresh 
saliva.  Triple  distilled  water  was  used  for  making  starch  disper- 
sions of  activators,  enzymes,  etc.,  and  for  rinsing  all  glassware. 

Method. — The  method7  of  testing  the  influence  of  the  amino  acids 
is  briefly  as  follows.  An  amount  of  air-dry  starch  equivalent  to  the 
required  amount  of  anhydrous  material  is  weighed  out,  mixed  with 
a  little  cold  water,  dispersed  by  pouring  into  boiling  water  (about 
80  c.c.  per  gm.  of  starch)  and  boiled  for  about  3  minutes.  This  is 

i  Sherman,   Kendall   and  Clark,   Jour.   Amer.   Chem.    Soc.,  32,    1082    (1910). 


upon  the  Enzymic  Hydrolysis  of  Starch  5 

transferred  to  100  cc.  cylinders,  neutralized  with  0.01  N  sodium 
hydroxide  solution  and  the  salts8  most  favorable  for  the  action  of 
the  amylase  added.  The  dispersions  are  then  made  up  to  100  c.c. 
so  that  the  concentration  of  starch  is  exactly  1  per  cent,  mixed 
thoroughly  by  stirring  and  placed  in  the  40°  bath  to  reach  the  desired 
temperature.  In  the  meantime,  the  enzyme  solution  is  prepared  and 
the  required  amount  pipetted  into  dry  flasks.  The  starch  dispersions 
are  then  poured  into  the  flasks  containing  the  enzyme  at  intervals 
of  15  seconds  and  the  flasks  placed  in  the  40°  bath.  At  the  end  of 
30  minutes,  enzymic  action  is  stopped  by  pouring  50  c.c.  of  Fehling 
solution  into  the  digestion  mixtures,  at  intervals  of  15  seconds  and 
in  the  same  order  in  which  the  starch  was  poured  on  the  enzyme. 
The  amount  of  reducing  sugar  formed  is  determined  by  immersing 
the  flasks  in  a  boiling  water  bath  for  15  minutes.  The  cuprous  oxide 
is  filtered  into  weighed  Gooch  crucibles,  washed  with  hot  water, 
alcohol  and  ether,  dried  at  100°,  and  weighed.  Glycine  and  alanine 
being  quite  soluble  were  dissolved  in  a  small  volume  of  water  and 
added  to  the  starch  paste,  after  it  was  poured  into  the  cylinders  and 
before  being  made  up  to  volume.  Since  tyrosine  and  phenylalanine 
are  difficultly  soluble,  the  amount  of  each  used  was  added  to  the 
water  in  which  the  starch  was  dispersed  and  boiled  with  it.  To  show 
whether  this  variation  in  procedure  affected  the  action  of  the  amino 
acid  on  the  enzyme,  digestions  were  carried  out  in  which  equal 
amounts  of  asparagine  were  added  before  boiling  in  some  cases  and 
after  cooling  in  others.  Activation  due  to  asparagine  was  the  same 
in  both  cases.  The  same  test  was  made  with  aspartic  acid  with  the 
same  result.  In  these  experiments  the  amino  acids  were  made 
neutral  to  rosolic  acid  with  0.01  N  sodium  hydroxide  solution. 

MEASUREMENT  OF  THE  INFLUENCE  OF  DIFFERENT 

AMINO  ACIDS 

Tables  I-IV  show  the  influence  of  carefully  neutralized  glycine, 
alanine,  tyrosine  and  phenylalanine,  added  separately  and  in  com- 
bination with  a  second  amino  acid,  upon  the  rate  of  hydrolysis  of 
"soluble  starch"  by  different  enzymes.  The  reducing  sugar  formed 

8  Sherman,  Thomas  and  Baldwin,  ibid.,  41,  231  (1919).  Pending  further  investigation 
the  substrate  is  prepared  in  the  same  manner  for  the  action  of  saliva  as  for  pancneatic 
amylase. 


6  Influence  of  Certain  Amino  Acids 

by  enzymic  hydrolysis  is  chiefly  maltose,  but  since  small  amounts 
of  glucose  may  also  be  present  the  results  are  stated  in  terms  of  the 
weight  of  cuprous  oxide  resulting  from  the  reduction  of  Fehling 
solution  by  the  sugar  or  sugars  present.  The  amounts  of  enzyme 
used  in  the  experiments  were  so  regulated  as  to  result  in  the  trans- 
formation of  about  Ys  of  the  starch  into  sugar. 

TABLE  I 

Effect  of  Glycine  and  Glycine  Plus  Aspartic  Acid  on  the  Enzymic  Hydrolysis 
of  Lintner  Soluble  Starch 


Amino  Acid,   Mg. 


Cuprous    Oxide,    Mg. 


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£ 

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277 

222 

292 

50            0 

280 

243 

334 

270 

286 

226 

292 

100             0 

284 

245 

341 

273 

283 

224 

292 

0           50 

279 

242 

344 

269 

280 

223 

289 

50           50 

279 

247 

334 

268 

281 

224 

292 

Activation  due 

to   glycine 

38 

18 

25 

13 

9 

4 

0 

TABLE  II 

Effect   of   Alanine   and  Alanine   Plus   Glycine   on   the   Ensymic   Hydrolysis 
of  Lintner  Soluble  Starch 


Amino  Acid,  Mg. 


Cuprous   Oxide,    Mg. 


u 

B 

V 

^    TO    (fl 

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II 

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273 

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248 

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310 

293 

339 

295 

251 

279 

282 

100 

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318 

301 

352 

301 

257 

287 

290 

0 

50 

320 

300 

355 

299 

249 

277 

280 

50 

50 

318 

296 

360 

305 

256 

284 

286 

25 

25 

317 

298 

351 

299 

250 

273 

283 

Activation  due 

to    alanine 

42 

20 

33 

16 

9 

15 

11 

upon  the  Ensymic  Hydrolysis  of  Starch  7 

TABLE  III 

Effect  of  Tyrosine  and  Tyrosine  Plus  Asparagine  on  the  Ensymic  Hydrolysis 
of  Lintner  Soluble  Starch 

Amino  Acid,   Mg.  Cuprous   Oxide,    Mg. 


"88s         s§5  w         'as       s~; 

£07.  >       |J         7!         s-5. 


0      0 

282 

294 

323 

248 

252 

261 

287 

50     0 

318 

317 

353 

262 

266 

273 

299 

0     50 

316 

312 

352 

256 

255 

263 

291 

25     25 

317 

317 

355 

263 

259 

269 

296 

50     50 

322 

319 

362 

263 

263 

270 

297 

100     0 

— 

— 

356 

— 

— 

— 

— 

0    100 

— 

— 

364 

— 

— 

— 

— 

Activation  due 

to  tyrosine 

36 

23 

30 

14 

14 

12 

12 

TABLE  IV 

Effect  of  Phenylalanine  and  Phenylalanine  Plus  Asparagine  on  the  Ensymic 
Hydrolysis  of  Lintner  Soluble  Starch 

Amino  Acid,   Mg.  Cuprous   Oxide,    Mg. 


— 

1 

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0 

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267 

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207 

244 

250 

252 

288 

50 

0 

300 

306 

222 

251 

253 

261 

290 

100 

0 

303 

307 

225 

256 

256 

263 

293 

0 

50 

309 

312 

225 

253 

254 

259 

291 

50 

50 

309 

312 

225 

257 

257 

264 

293 

25 

25 

308 

311 

225 

260 

255 

261 

292 

Activation  due 
to  phenylalanine      36  14  18  12  6  11  5 

The  data  given  in  the  above  tables  show  an  undoubted  increase 
in  the  activity  of  purified  pancreatic  amylase,  pancreatin,  saliva,  and 
purified  malt  amylase  in  the  presence  of  any  one  of  the  four  amino 
acids  investigated  or  of  any  two  of  them  whose  joint  effects  were 
tested.  The  apparent  activation  is  not  so  marked  in  case  of  the  less 


8  Influence   of  Certain  Ammo   Acids 

sensitive  enzymes,  malt  extract,  takadiastase  and  aspergillus  amylase. 
It  is  also  true  that  the  acceleration  of  hydrolysis  by  the  amino  acids 
is  somewhat  greater  for  the  purified  form  of  the  enzyme  than  for 
the  natural  or  commercial  material  in  which  the  enzyme  is  accom- 
panied by  other  constituents  of  the  tissue  or  secretion  in  question. 
It  will  be  observed  that,  in  general,  the  four  amino  acids  here  dis- 
cussed as  well  as  asparagine  and  aspartic  acid  previously  studied9 
behave  in  a  similar  manner.  The  above  results  show  no  evidence  that 
the  addition  of  two  amino  acids  to  the  same  digestion  mixture  causes 
greater  activation  than  would  result  from  a  corresponding  concentra- 
tion of  one  of  them.  The  following  combinations  have  been  tested : 
aspartic  acid  and  asparagine,  glycine  and  aspartic  acid,  tyrosine  and 
asparagine,  phenylalanine  and  asparagine,  alanine  and  glycine. 

Since  some  investigators  have  held  that  the  activating  effect  of 
amino  acids  is  attributable  to  their  presence  inducing  a  more  favorable 
hydrogen  ion  concentration  in  the  digestion  mixture,  we  have  deter- 
mined electrometrically  the  hydrogen  ion  concentrations  of  our  mix- 
tures with  and  without  neutralized  amino  acid,  with  the  results  shown 
in  Table  V.  It  is  evident  that  the  reaction  of  our  mixtures  is  not 
changed  by  the  addition  of  the  neutralized  amino  acids  to  any  signi- 
ficant degree  and  therefore  that  the  favorable  effect  of  the  amino 
acid  upon  the  enzyme  action  is  due  to  some  other  cause  or  causes. 

TABLE  V 

Hydrogen    Ion    Concentration   in    Solutions   with    and    without   Neutralized 

Amino  Acids 

Solj 
Amino  Acid 

None 

50  mg.  glycine 

50  mg.  alanine 

50  mg.  tyrosine 

50  mg.  phenylalanine 

MODE  OF  ACTION  OF  THE  AMINO  ACID 

Several  possible  explanations  of  the  favorable  influence  of  the 
amino  acids  may  be  suggested.  (1)  There  may  be  a  direct  accel- 

8  Sherman  and  Walker,  Jour.  Amer.  Chem.   Soc.,  41,   1867   (1919). 


Soln.   activated 
as  for  pancreatic 
amylase 

.  Soln.   activated 
as    for    malt 
amylase 

Soln.    activated 
as  for  aspergillus 
amylase 

PH+ 

PH+ 

PH+ 

6.90 

4.46 

4.91 

6.86 

4.50 

5.01 

6.88 

4.45 

4.91 

6.86 

4.46 

4.89 

6.88 

4.48 

4.91 

upon  the  Enzymic  Hydrolysis  of  Starch  9 

crating  effect  upon  the  enzyme-starch  reaction.  (2)  The  amino  acid 
may  combine  with  one  or  more  products  of  digestion  which,  if  free, 
might  retard  the  enzyme  action.  (3)  The  amino  acid  may  protect  the 
enzyme  against  the  deleterious  influence  of  some  accidental  or 
unknown  impurity.  (4)  The  amino  acid  may  retard  hydrolytic 
destruction  of  the  enzyme. 

Is  the  Action  Direct? — It  is  conceivable  that  the  amino  acid  may 
directly  facilitate  the  interaction  of  the  enzyme  and  substrate.  Until 
our  knowledge  of  the  mechanism  of  enzyme  action  is  further  de- 
veloped this  suggestion  can  only  be  approached  by  somewhat 
speculative  discussion,  or  experimentally  by  a  process  of  elimination 
of  other  possibilities. 

Does  the  Effect  Depend  upon  Some  Reaction  with  the  Products 
of  Digestion? — Since  the  activity  of  an  enzyme  is  often  diminished 
by  the  accumulation  of  the  products  of  its  action,  it  might  be  sug- 
gested that  the  amino  acids  exert  their  favorable  influence  through 
combining  with  some  product  or  products  of  the  hydrolysis  which 
might  otherwise  combine  with  the  enzyme  itself  thus  reducing  its 
activity,  or  it  might,  if  remaining  free  in  the  solution,  tend  to  bring 
the  hydrolysis  to  equilibrium.  To  test  this  point,  the  effect  of  the 
addition  of  100  mg.  of  pure  maltose  to  the  starch  paste,  with  and 
without  glycine,  on  hydrolysis  by  pancreatic  amylase  was  determined. 
Similar  experiments  in  which  a  certain  amount  of  a  hydrolytic 
mixture  was  substituted  for  pure  maltose  were  carried  out  as  follows. 
One  gm.  of  starch  was  digested  for  1  hour  at  40°C.  by  pancreatic 
amylase  preparation,  at  the  end  of  which  time  the  enzyme  was 
destroyed  by  boiling.  The  effect  of  25  and  50  c.c.  of  this  digested 
mixture  on  hydrolysis  with  and  without  glycine  was  tested.  The  fact 
that  known  amounts  of  sodium  chloride  and  phosphate  were  added 
with  the  digested  material  was  taken  into  consideration,  and  the 
volumes  of  these  activators  added  to  the  substrates  containing  the 
hydrolytic  mixtures  were  adjusted  accordingly.  Correction  being 
made  for  their  reducing  power,  both  pure  maltose  and  the  hydrolytic 
products  of  the  starch  were  found  under  the  conditions  of  these 
experiments  to  be  without  measurable  effect  upon  the  activity  of  the 
enzyme  used,  showing  that  the  favorable  influence  of  the  amino  acid 
cannot  be  explained  in  this  way. 


10  Influence   of  Certain  Ammo   Acids 

Does  the  Amino  Acid  Protect  the  Enzyme  against  Some  Accidental 
or  Unknown  Deleterious  Influence? — Aside  from  the  possibility  of 
correcting  an  unfavorable  hydrogen  ion  concentration  which  has 
already  been  excluded  as  an  explanation  of  our  results,  it  is  possible 
that  the  amino  acid  may  act  by  protecting  the  enzyme  from  some 
active  but  unknown  deleterious  influence.  This  is  illustrated  by  the 
following  experiments  with  cupric  sulphate. 

Protective  Action  of  Amino  Acids  against  Cupric  Sulphate. — 
These  experiments  were  designed  to  show  whether  the  deleterious 
effect  upon  amylase  activity  of  such  a  heavy  metal  salt  as  copper 
sulphate  could  be  wholly  or  in  part  overcome  by  the  presence  of  an 
amino  acid.  In  the  experiments  the  results  of  which  are  given  in 
Table  VI,  the  cupric  sulphate  and  amino  acid  were  added  to  the 
cooled  starch  paste  and  thoroughly  mixed  before  pouring  onto  the 
enzyme  solution. 

TABLE  VI 

Action  of  Amino  Acids  in  Protecting  Purified  Pancreatic  Amylase  from  the 
Deleterious  Effect  of  Copper 

Amino  Acid  Mg.  Cone.    CuSOi   in  Cuprous    oxide 

starch   paste  Mg. 

None                                                                             0  274 

None  0.00003  M  58 

Alanine  100  0.00003  M  298 

Asparagine  100  0.00003  M  293 

Glycine  100  0.00003  M  304 

Glycine  50  0.00003  M  295 

Glycine  100                                    0  308 

The  above  data  show  that  a  0.00003  M  concentration  of  cupric 
sulphate  in  the  digestion  mixture  diminished  the  activity  of  pan- 
creatic amylase  by  about  78  per  cent.  This  is  in  accordance  with 
results  recently  obtained  in  this  laboratory  by  Sherman  and  Wayman.10 
However,  upon  the  addition  of  0.1  per  cent  of  amino  acid,  not  only 
is  the  inhibiting  influence  of  the  cupric  sulphate  counteracted,  but 
there  is  an  increase  in  saccharification  almost  equal  to  that  which 
occurs  in  the  presence  of  amino  acid  and  absence  of  copper.  Further 
experiments  were  performed  in  which  the  cupric  sulphate  was  added 
directly  to  the  enzyme  solution  in  two  concentrations,  0.0035  M  and 
0.00003  M  and  the  efficiency  of  0.1  per  cent  glycine  in  the  substrate 
in  reactivating  the  enzyme  was  studied.  Some  results  are  given  in 
Table  VII. 

10  Jour.  Amer.   Chem.    Soc.,  43,   2454    (1921). 


upon  the  Enzymic  Hydrolysis  of  Starch  11 

TABLE  VII 
Reactivation  of  Pancreatic  Amylase  by  Glycine  after  Inactivation  by  Copper 

_  o 

.s  .sg  -ss 

QV  o*3  O-g 

i              CAM  Wo  ««  R 

fl              s  g.  »  M  ,=?  c                         Cuprous    oxide 

J5>           "  S                                -2 

5  O  "5  0°    E  O    « 

gJ3  g|  §8  Exp.l        Exp.2       Exp.3 

Mg.        uto  U§  U-5  Mg.  Mg.  Mg. 

00  0  0  280  252  264 

100  0  0  0  316  —  300 

100  0  0.0035  M  0.00003  M  98  —  — 

0  0  0.0035  M  0.00003  M  4  —  — 

0  0  0.00003  M  0.00000018  M  —  209  

100  0  0.00003  M  0.00000018  M  —  260  184 

100  0.00003  M  0.00003  M  0.00003  M  260  182 

Numerous  experiments  showed  that  the  inactivation  of  the  enzyme 
by  the  copper  and  its  reactivation  by  amino  acid  were  considerably 
influenced  by  time  and  temperature.  In  Expt.  2  of  Table  VII  the 
solution  stood  for  12  minutes  before  testing;  in  Expt.  3  it  stood  for 
55  minutes.  The  influence  of  temperature  is  illustrated  in  Table  VIII. 

TABLE  VIII 

Effect  of   Temperature   on  Reactivation  of  Pancreatic  Amylase   by   Glycine 
after  standing  for  20  minutes  in  0.00003  M  cupric  sulphate  solution 

Glycine  Temp,    of    enzyme    sol.  Cuprous  oxide 

Mg.  after  20   min.  Mg. 

0  23°C  115 

100  23  142 

0  12  175 

100  12  213 

As  to  the  bearing  of  these  experiments  with  cupric  sulphate  upon 
the  question  whether  the  role  of  amino  acids  is  that  of  a  direct 
accelerator  of  the  enzyme  action  or  rather  that  of  a  protector  which 
increases  the  amount  of  work  done  by  the  enzyme  through  preventing 
its  deterioration,  it  cannot  be  doubted  that  they  establish  the  pos- 
sibility of  a  very  marked  protective  effect  without  precluding  the 
additional  possibility  of  a  more  direct  action  upon  the  enzyme. 

Evidently  with  a  low  concentration  of  copper  ions  in  solution  these 
react  with  amino  acid  forming  copper-amino  ions11  more  readily  than 
with  the  enzyme;  and  moreover,  when  the  copper  ion  has  already 

11 J.  T.  Barker,  Trans.   Faraday   Soc.,  3,   188   (1908). 


12  Influence  of  Certain  Amino  Acids 

acted  upon  and  inactivated  a  part  of  the  enzyme,  it  apparently  may 
still  be  taken  up  by  the  amino  acid,  and  the  enzyme  thus  freed  from 
the  copper  may  become  active  again. 

Does  the  Amino  Acid  Act  by  Retarding  the  Hydrolytic  Destruc- 
tion of  the  Enzyme? — Another  possibility  is  that  the  amino  acid  may 
act  by  preventing  or  retarding  the  deterioration  of  the  enzyme  in  its 
aqueous  solution.  The  very  rapid  deterioration  of  water  solutions 
of  pancreatic  amylase,  particularly  when  highly  purified,  and  the 
influence  of  the  sodium  chloride  and  secondary  phosphate  (regularly 
used  to  "activate"  this  enzyme)  in  retarding  the  deterioration  have 
been  discussed  in  previous  papers  from  this  laboratory.12  Since  the 
deterioration  of  enzymic  activity,  while  greatly  retarded,  is  not 
entirely  prevented  by  the  presence  of  the  salts,  it  is  not  improbable 
that  the  favorable  influence  of  the  amino  acid  may  be  due  at  least 
in  part  to  a  further  protection  of  the  enzyme  from  deterioration  in 
the  aqueous  dispersion  in  which  it  acts.  In  studying  this  point  three 
methods  of  investigation  have  been  used.  (1)  Enzyme  solutions 
with  and  without  amino  acid  were  allowed  to  stand  for  a  definite 
length  of  time  at  known  temperatures  in  absence  of  substrate  and 
the  resulting  loss  of  activity  compared.  (2) The  effect  upon  activa- 
tion due  to  amino  acids  of  varying  the  temperature  at  which  the 
enzyme  was  allowed  to  act  was  determined.  (3)  A  similar  series 
of  experiments  was  carried  out  in  which  the  length  of  time  of  diges- 
tion as  well  as  the  temperature  was  varied. 

The  method  of  determining  the  effect  of  an  amino  acid  in  retarding 
the  deterioration  of  purified  pancreatic  amylase  in  water  solution 
was  as  follows.  10  mg.  of  enzyme  were  dissolved  in  100  c.c.  of  water 
at  ice  box  temperature  containing  5  c.c.  M  NaCl  and  2.5  c.c.  M/50 
Na2HPO4.  10  c.c.  were  pipetted  into  each  of  four  25  c.c.  beakers, 
the  second  of  which  contained  20  mg.  of  alanine.  At  intervals  of 
eight  minutes  20  mg.  of  alanine  were  added  to  the  third  and  fourth 
beakers.  None  was  added  to  enzyme  solution  in  fourth  beaker. 
These  stood  at  room  temperature  for  33  minutes  after  the  last  addi- 
tion of  alanine.  Then  0.6  c.c.  from  each  beaker  was  pipetted  into 
flasks  and  substrate  poured  on  and  digestions  carried  out  as  usual. 
The  substrates  digested  by  the  amylase  solutions  to  which  the  amino 
acid  had  been  added  contained  1.2  mg.  alanine  put  in  with  0.6  c.c. 

12  Sherman    and    Schlesinger,    Jour.    Amer.    Chem.    Soc.    Series    of    papers    (1912-1916). 


upon  the  Enzymic  Hydrolysis  of  Starch  13 

of  enzyme,  therefore,  to  equalize  conditions,  1.2  mg.  were  added  to 
starch  dispersions  acted  upon  by  the  control  solution.  When  tested 
the  solutions  to  which  alanine  had  not  been  added  were  as  active  as 
those  containing  0.2  per  cent  of  the  amino  acid. 

The  procedure  was  then  varied,  the  enzyme  being  allowed  to  stand 
at  two  different  temperatures  in  solutions  containing  salt,  phosphate 
and  alanine  in  different  combinations.  Results  are  shown  in  Table 
IX.  In  experiments  1  and  2,  indicated  in  following  table,  the 
temperature  was  accurately  controlled  at  22°C.,  in  experiment  3, 
at  40°C. 

TABLE  IX 

Effect   of  0.1   Per   Cent   Alanine   in   Retarding   Deterioration   of   Pancreatic 
Amylase  in  Solution  at  22°C  and  40°C. 

Alanine  NaCl  Na2HPO4  Cuprous    Oxide,    Mg. 

(0.1%)  (optimum   cone.)  (optimum   cone.)  Exp.  1         Exp.  2          Exp.  3 

22°C  22°C  40°C 

absent  present  present  274  144 

present  present  present  275  198 

present  absent  absent  133 

absent  absent  absent  98 

absent  present  absent  273 

present  present  absent  272 

absent  absent  present  238 

present  absent  present  269 

The  above  table  indicates  that  the  amino  acid  does  retard  the 
hydrolytic  destruction  of  the  amylase.  Solutions  of  pancreatic 
amylase  (containing  optimum  concentrations  of  sodium  chloride  and 
phosphate)  which  have  stood  1  hour  at  40° C.  show  about  y$  greater 
amylolytic  activity  when  alanine  has  been  added  to  the  solution  in 
advance.  Under  similar  conditions  except  that  the  solution  is  kept 
at  22°C.  instead  of  40°C.  there  is  no  measurable  difference,  probably 
because  the  deterioration  at  this  temperature  in  the  presence  of 
optimum  concentrations  of  salts  is  small  in  either  case.  It  is  probable 
that  at  22°C.  the  amino  acid  has  little  or  no  effect  on  the  rate  of 
hydrolysis  of  starch  by  amylase,  since,  as  will  be  shown  later,  the 
activation  decreases  markedly  with  temperature.  It  further  appears 
from  Table  IX  that  amylase  solutions  to  which  the  usual  NaCl  and 
Na2HPO4  have  not  been  added  are  protected  by  alanine  even  at  22°. 
At  this  temperature  the  amino  acid  is  apparently  capable  of  replacing 
either  the  sodium  chloride  or  the  phosphate  without  altering  the 


14  Influence   of  Certain  Amino  Acids 

activity  of  the  solution,  but  not  both  salts.  In  these  experiments, 
however,  the  conditions  were  such  as  to  result  in  greater  deterioration 
than  occurs  in  our  ordinary  tests  of  enzyme  activity  both  because  of 
longer  exposure  of  the  enzyme  to  water  and  because  of  the  absence 
of  its  substrate. 

If  protection  against  hydrolytic  destruction  is  a  partial  explanation 
of  the  increased  activity  of  enzymes  in  the  presence  of  amino  acids, 
it  would  be  logical  to  expect  that  any  condition  favoring  the  hydrolysis 
of  the  enzyme  molecule,  such  as  a  higher  temperature  or  subjection 
to  a  given  temperature  for  a  longer  period,  would  cause  greater  ap- 
parent activation  by  the  amino  acid.  To  test  this  point,  a  series  of 
experiments  was  planned  in  which  30  minute  and  60  minute  digestions 
by  pancreatic  amylase  with  and  without  amino  acid  were  carried  out 
at  temperatures  ranging  from  30°-75°C.  Table  X  shows  the  results 
obtained  with  glycine  at  different  temperatures  when  0.6  c.c.  of 
0.01  per  cent  enzyme  solution  acts  upon  100  c.c.  of  substrate  for 
30  minutes  and  when  one-half  the  amount  acts  upon  the  same  amount 
of  substrate  for  60  minutes.  The  experiments  were  repeated  with 
phenylalanine  replacing  glycine,  the  results  of  which  are  given  in 
Table  XI.  The  purified  pancreatic  amylase  preparation  employed 
in  the  phenylalanine  experiments  was  about  a  year  older  than  that 
used  with  glycine  and  only  two-thirds  as  active,  consequently,  one- 
third  more  of  the  solution  was  added  to  each  digestion  mixture. 

TABLE  X 

Effect   of   Variation   of   Temperature  on  Activation   of  Purified  Pancreatic 

Amylase  by  Glycine 

30    minute    digestions  60    minute   digestions 

Cuprous  Oxide,    Mg.  Cuprous  Oxide,    Mg. 


30°C 

40° 

50° 

55° 

57° 

60° 

65° 

66.5° 

70° 

75° 


"ei                  Activation 

B|| 

"bo                   Activation 

•^  & 

o 

due  to 

50  mg. 

o 

due 

to  50  mg. 

"3>°- 

fc 

glycine 

10  "So  o. 

glycine 

155 

136 

19 

14% 

142 

114 

28 

24% 

252 

220 

32 

14% 

287 

248 

39 

12% 

366 

311 

55 

17% 

343 

259 

84 

32% 

393 

291 

102 

35% 

369 

214 

155 

72% 

363 

251 

112 

45% 

367 

167 

200 

120% 

380 

183 

197 

107% 

320 

84 

236 

281% 

176 

58 

118 

203% 

99 

24 

75 

312% 

110 

30 

80 

266% 

52 

19 

33 

173% 

55 

24 

31 

129% 

18 

8 

10 

125% 

0 

0 

0 

0% 

0 

0 

0 

0% 

upon  the  Enzymic  Hydrolysis  of  Starch  15 

TABLE  XI 

Effect  of   Variation   of   Temperature   on  Activation   of  Purified  Pancreatic 
Amylase  by  Phenylalanine 

30    minute    dijjest'ons  60  minute   digestions 

Cuprous  Oxide,   Mg.  Cuprous  Oxide,   Mg. 


Ill 

o-S                   Activation 

a>»  C 
C  ""* 

•S..S 

Activation 

s8 

o  a 

due  to 

50  mg. 

«j  S 

o  c 

due  to 

50  me. 

°-S"3 

m  o<  rt 

^  —  ^ 

phenylalanine           S'S'3            ^"a 

phenylalanine 

183 

164 

19 

12% 

173 

143 

30 

21% 

291 

258 

33 

12% 

290 

247 

43 

17% 

340 

255 

85 

33% 

300 

194 

106 

55% 

291 

181 

110 

60% 

226 

94 

132 

140% 

134 

66 

68 

103% 

105 

37 

68 

184% 

42 

25 

17 

68% 

22 

12 

10 

83% 

10 

7 

3 

43% 

6 

4 

2 

50% 

0 

H 

30°  C 

40° 

50° 

55° 

60° 

65° 

70° 

The  results  of  the  above  experiments  afford  striking  evidence 
that  deterioration  of  the  enzyme  with  increase  in  temperature  is 
retarded  by  amino  acids.  For  the  30  minute  digestions,  beginning 
with  an  increase  at  30°C.  of  19  mg.  of  cuprous  oxide  or  14  per  cent, 
the  activating  effect  of  glycine  reaches  a  maximum  of  197  mg.  of 
cuprous  oxide  or  over  100  per  cent  at  60°C.  Above  this  temperature 
the  acceleration  as  represented  by  increase  in  mg.  of  cuprous  oxide 
declines  sharply,  although  the  percentage  activation  continues  to  in- 
crease up  to  66.5°C.  The  rapid  falling  off  in  activity  after  60°C.  is 
doubtless  due  to  coagulation  of  the  amylase  which  is  not  prevented  by 
the  amino  acid. 

The  experiments  with  phenylalanine  show  the  same  general  effect 
though  not  as  marked  as  in  the  case  of  glycine.  Maximum  activation 
occurs  at  55°C.  instead  of  60°C.  after  which  the  decline  is  rapid. 
It  will  be  observed  from  the  tables  that  the  two  amylase  preparations 
in  absence  of  either  amino  acid  behave  quite  differently,  the  less 
active  one,  employed  in  connection  with  phenylalanine,  being  destroyed 
more  rapidly  with  increasing  temperature.  Glycine  added  to  a  sub- 
strate hydrolyzed  by  this  enzyme  at  60°C.  gave  practically  the  same 
result  as  obtained  with  phenylalanine.  Therefore,  it  appears  that 
the  lack  of  agreement  in  the  results  is  due  not  to  dissimilarity  in 
the  action  of  the  two  amino  acids  but  rather  to  some  difference  in 
the  amylases,  probably  connected  with  the  deterioration  which  had 
already  occurred  in  the  less  active  preparation. 


16  Influence   of   Certain  Amino.   Acids 

The  most  evident  explanation  of  this  marked  temperature  effect 
is  that  the  amino  acids  preserve  the  enzyme  in  solution  from  the 
destructive  influence  of  heat.  In  so  far  as  the  result  of  enzymic 
hydrolysis  is  concerned,  increase  in  temperature  exerts  two  opposite 
influences  upon  amylolytic  action.  It  accelerates  the  velocity  of 
conversion  of  starch  into  sugar  and  at  the  same  time  increases  the 
rate  of  deterioration  of  the  enzyme.  The  second  reaction  being 
retarded  by  the  presence  of  one  of  the  decomposition  products,  the 
first  effect,  that  is,  increase  in  the  rate  of  hydrolysis  of  the  starch, 
becomes  more  noticeable. 

When  hydrolysis  continued  for  60  minutes  the  amino  acids  pro- 
duced a  greater  apparent  activation  at  all  temperatures  until  after 
coagulation  of  the  enzyme  had  occurred  than  was  observed  for  the 
shorter  period  digestions.  Digestions  carried  out  at  40° C.  for  periods 
of  time  from  20  minutes  to  3  hours  with  and  without  glycine  and 
tyrosine  show  the  same  increase  in  apparent  activation  with  length 
of  time  of  action  of  the  amylase.  This  is  what  would  be  expected 
if  the  amino  acid  activates  by  protecting  against  deterioration,  since 
the  longer  the  enzyme  is  subjected  to  an  injurious  temperature,  the 
greater  the  deterioration  and  consequently  the  more  marked  the 
activating  effect  in  digestion  mixtures  in  which  the  destruction  is 
partially  prevented. 

The  fact,  demonstrated  by  the  above  data,  that  the  presence  of 
certain  amino  acids  retards  the  deterioration  of  the  enzyme  consti- 
tutes an  interesting  addition  to  the  evidence  supporting  the  view  that 
the  enzyme  itself  is  a  substance  of  protein  nature  or  which  contains 
protein  as  an  essential  constituent. 

SUMMARY 

Addition  of  glycine,  alanine,  phenylalanine  or  tyrosine  caused  an 
undoubted  increase  in  the  rate  of  hydrolysis  of  starch  by  purified 
pancreatic  amylase,  commercial  pancreatin,  saliva  or  purified  malt 
amylase.  Less  marked  results  were  obtained  with  the  less  sensitive 
enzyme  materials,  malt  extract,  takadiastase  and  aspergillus  amylase. 

Each  of  the  four  amino  acids  here  studied  as  well  as  aspartic  acid 
and  asparagine  previously  investigated,  showed  a  similar  favorable 
influence  upon  the  enzymic  hydrolysis  of  starch. 


upon  the  Enzymic  Hydrolysis  of  Starch  17 

The  addition  of  a  mixture  of  two  amino  acids  produced  no  greater 
effect  than  would  result  from  the  same  concentration  of  one  of  them. 

In  these  experiments  the  favorable  effect  of  the  added  amino  acid 
was  not  due  to  any  influence  upon  hydrogen  ion  concentration  nor 
to  combination  of  the  amino  acid  with  the  products  of  the  enzyme 
reaction. 

The  addition  of  one  of  these  amino  acids  is  a  very  effective  means 
of  protecting  the  enzyme  from  the  deleterious  influence  of  cupric 
sulphate  and  may  even  serve  to  restore  to  full  activity  an  enzyme 
which  has  been  partially  inactivated  by  copper. 

The  favorable  influence  of  the  amino  acid  is  evidently  due  in  part 
at  least  to  a  protection  of  the  enzyme  from  deterioration  in  the  aqueous 
solution  in  which  it  acts.  This  view  is  supported  by  the  following 
facts:  (1)  Solutions  of  pancreatic  amylase  (containing  optimum 
concentrations  of  chloride  and  phosphate)  which  have  stood  1  hour 
at  40° C.  show  ^  greater  activity  when  alanine  has  been  added  to 
the  solution  in  advance.  Amylase  solutions  to  which  the  chloride  and 
phosphate  have  not  been  added  are  protected  by  alanine  at  22 °C. 
(2)  There  is  a  striking  increase  in  activation  by  glycine  and  phenyl- 
alanine  with  increased  temperature  until  coagulation  of  the  enzyme 
occurs.  (3)  At  the  same  temperature,  there  is  greater  apparent 
activation  when  hydrolysis  is  allowed  to  proceed  for  1  hour  than 
when  the  action  is  stopped  at  the  end  of  30  minutes. 

This  explanation  of  the  mode  of  action  of  amino  acid  does  not 
preclude  the  possibility  of  a  more  direct  influence  upon  the  activity 
of  the  enzyme. 


VITA 

Florence  Walker  was  born  at  Oriole,  Md.,  October  15,  1887.  She 
was  prepared  for  college  in  the  Blackstone  School  for  Girls,  Black- 
stone,  Va.  Entering  Randolph-Macon  Woman's  College  in  1905 
she  received  the  degree  of  Bachelor  of  Arts  in  1909.  Until  1917  she 
was  Instructor  in  Chemistry  in  Randolph-Macon.  During  the  years 
1917-18  and  1921-22  and  Summer  Sessions  1916  and  1917  she 
pursued  the  study  of  chemistry  in  Columbia  University,  receiving 
the  degree  of  Master  of  Arts  in  1918.  From  1918  to  1921  she  was 
Carnegie  Research  Assistant  to  Professor  H.  C.  Sherman,  during 
which  period  she  was  co-author  with  Professor  Sherman  of  several 
papers  on  amylases.  For  the  past  year  she  has  held  a  University 
Fellowship  in  Chemistry. 


—TX.A,  BELow11"1831  DATE 


BIOLOGY 


LIBRARY" 


A78719 


